GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1''s central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.     

Glycogen synthase kinase-3beta (GSK3beta) negatively regulates PTTG1/human securin protein stability, and GSK3beta inactivation correlates with securin accumulation in breast tumors

PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates securin to promote its proteolysis via SCF(βTrCP) E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for securin accumulation in breast cancers.     

βTrCP-mediated ubiquitylation regulates protein stability of Mis18β in a cell cycle-dependent manner

Ubiquitin E3 ligases including SCF complex are key regulators of cell cycle. Here, we show that Mis18β, a component of Mis18 complex governing CENP-A localization, is a new substrate of βTrCP-containing SCF complex. βTrCP interacted with Mis18β exclusively during interphase but not during mitosis and mediated proteasomal degradation of Mis18β leading to the inactivation of Mis18 complex during interphase. In addition, uncontrolled stabilization of Mis18β caused cell death. Together, we propose that βTrCP-mediated regulation of Mis18β stability is a mechanism to restrict centromere function of Mis18 complex from late mitosis to early G1 phase.     

Ability of CK2β to selectively regulate cellular protein kinases

The Wee1 protein kinase plays a prominent role in keeping cyclin dependent kinase 1 (CDK1) inactive during the G2 phase of the cell cycle. At the onset of mitosis, Wee1 is ubiquitinated by the E3 ubiquitin ligase SCF(beta-TrCP) and subsequently degraded by the proteasome machinery. Previously, it has been reported that although Wee1 lacks the conserved binding motif recognised by beta-TrCP, the CDK-catalysed phosphorylation of Wee1 at Ser123 creates a phosphodegron and primes phosphorylation of two other protein kinases, polo-like kinase 1 (PLK1) and protein kinase CK2, which create two additional phosphodegrons recognised by beta-TrCP. These events contribute to destabilise Wee1 at the onset of mitosis (Watanabe et al. Proc Natl Acad Sci USA 101:4419-4424, 2004). We show here that in addition to the ability of CK2 to phosphorylate Wee1 as reported earlier, the regulatory beta-subunit of protein kinase CK2 can interact with Wee1 in high molecular mass complexes. Indirect immunofluorescence microscopy revealled subcellular co-localisation of CK2beta and Wee1 in the nucleus. Moreover, in vitro phosphorylation assays showed that CK2beta indirectly up-regulates the activity of CDK1 with respect to histone H1 phosphorylation by inhibiting Wee1 kinase. These findings support the view that CK2beta regulates various intracellular processes by modulating the activity of protein kinases that are distinct from CK2 and that protein kinase CK2 plays an important role in events related to the regulation of cell cycle progression as a tetrameric enzyme but also through the individual subunits.     

Monitoring the Cell Cycle by Multi-Kinase-Dependent Regulation of Swe1/Wee1 in Budding Yeast

In eukaryotes, G2/M transition is induced by the activation of cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. Recent advances in our understanding of mitotic entry in budding yeast has revealed that these cells utilize the level of Swe1 (Wee1 ortholog) phosphorylation as a means of monitoring cell cycle progression and of coordinating morphogenetic events with mitotic entry. Swe1 is phosphorylated by at least three distinct kinases at different stages of the cell cycle. This cumulative phosphorylation leads to the hyperphosphorylation and degradation of Swe1 through ubiquitin-mediated proteolysis. Thus, Swe1 functions as an important cell cycle modulator that integrates multiple upstream signals from prior cell cycle events before its ultimate degradation permits passage into mitosis.     

A G2-phase microtubule-damage response in fission yeast

Microtubules assume a variety of structures throughout the different stages of the cell cycle. Ensuring the correct assembly of such structures is essential to guarantee cell division. During mitosis, it is well established that the spindle assembly checkpoint monitors the correct attachment of sister chromatids to the mitotic spindle. However, the role that microtubule cytoskeleton integrity plays for cell-cycle progression during interphase is uncertain. Here we describe the existence of a mechanism, independent of the mitotic checkpoint, that delays entry into mitosis in response to G(2)-phase microtubule damage. Disassembly of the G(2)-phase microtubule array leads to the stabilization of the universal mitotic inhibitor Wee1, thus actively delaying entry into mitosis via inhibitory Cdc2 Tyr15 phosphorylation.     

Activation Domain-dependent Degradation of Somatic Wee1 Kinase

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G₂ and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.     

Cell cycle regulation of a Xenopus Wee1-like kinase.

Using a polymerase chain reaction-based strategy, we have isolated a gene encoding a Wee1-like kinase from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr-15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase.     

Molecular imaging of glycogen synthase kinase-3β and casein kinase-1α kinases

Glycogen synthase kinase-3β (GSK3β) and casein kinase-1α (CK1α) are multifunctional kinases that play critical roles in the regulation of a number of cellular processes. In spite of their importance, molecular imaging tools for noninvasive and real-time monitoring of their kinase activities have not been devised. Here we report development of the bioluminescent GSK3β and CK1α reporter (BGCR) based on firefly luciferase complementation. Treatment of SW620 cells stably expressing the reporter with inhibitors of GSK3β (SB415286 and LiCl) or CK1α (CKI-7) resulted in dose- and time-dependent increases in BGCR activity that were validated using Western blotting. No increase in bioluminescence was observed in the case of S37A mutant (GSK3β inhibitors) or S45A mutant (CKI-7), demonstrating the specificity of the reporter. Imaging of mice tumor xenograft generated with BGCR-expressing SW620 cells following treatment with LiCl showed unique oscillations in GSK3β activity that were corroborated by phosphorylated GSK3β immunoblotting. Taken together, the BGCR is a novel molecular imaging tool that reveals unique insight into GSK3β and CK1α kinase activities and may provide a powerful tool in experimental therapeutics for rapid optimization of dose and schedule of targeted therapies and for monitoring therapeutic response.     

Haspin inhibition delays cell cycle progression through interphase in cancer cells

Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or the S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby, indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis.     

14-3-3 binding regulates catalytic activity of human Wee1 kinase.

The mitotic inducer Cdc2 is negatively regulated, in part, by phosphorylation on tyrosine 15. Human Wee1 is a tyrosine-specific protein kinase that phosphorylates Cdc2 on tyrosine 15. Human Wee1 is subject to multiple levels of regulation including reversible phosphorylation, proteolysis, and protein-protein interactions. Here we have investigated the contributions made by 14-3-3 binding to human Wee1 regulation and function. We report that the interactions of 14-3-3 proteins with human Wee1 are reduced during mitosis and are stable in the presence of the protein kinase inhibitor UCN-01. A mutant of Wee1 that is incapable of binding to 14-3-3 proteins has lower enzymatic activity, and this likely accounts for its reduced potency relative to wild-type Wee1 in inducing a G(2) cell cycle delay when overproduced in vivo. These findings indicate that 14-3-3 proteins function as positive regulators of the human Wee1 protein kinase.     

Glycogen synthase kinase 3 phosphorylates RBL2/p130 during quiescence

Phosphorylation of the retinoblastoma-related or pocket proteins RB1/pRb, RBL1/p107, and RBL2/p130 regulates cell cycle progression and exit. While all pocket proteins are phosphorylated by cyclin-dependent kinases (CDKs) during the G1/S-phase transition, p130 is also specifically phosphorylated in G0-arrested cells. We have previously identified several phosphorylated residues that match the consensus site for glycogen synthase kinase 3 (GSK3) in the G0 form of p130. Using small-molecule inhibitors of GSK3, site-specific mutants of p130, and phospho-specific antibodies, we demonstrate here that GSK3 phosphorylates p130 during G0. Phosphorylation of p130 by GSK3 contributes to the stability of p130 but does not affect its ability to interact with E2F4 or cyclins. Regulation of p130 by GSK3 provides a novel link between growth factor signaling and regulation of the cell cycle progression and exit.     

Inhibition of S/G2 phase CDK4 reduces mitotic fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.     

Two distinct mechanisms for negative regulation of the Wee1 protein kinase.

The Wee1 protein kinase negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein. To examine the potential mechanisms for Wee1 regulation during the cell cycle, we have introduced a recombinant form of the fission yeast Wee1 protein kinase into Xenopus egg extracts. We find that the Wee1 protein undergoes dramatic changes in its phosphorylation state and kinase activity during the cell cycle. The Wee1 protein oscillates between an underphosphorylated 107 kDa form during interphase and a hyperphosphorylated 170 kDa version at mitosis. The mitosis-specific hyperphosphorylation of the Wee1 protein results in a substantial reduction in its activity as a Cdc2-specific tyrosine kinase. This phosphorylation occurs in the N-terminal region of the protein that lies outside the C-terminal catalytic domain, which was recently shown to be a substrate for the fission yeast Nim1 protein kinase. These experiments demonstrate the existence of a Wee1 regulatory system, consisting of both a Wee1-inhibitory kinase and a Wee1-stimulatory phosphatase, which controls the phosphorylation of the N-terminal region of the Wee1 protein. Moreover, these findings indicate that there are apparently two potential mechanisms for negative regulation of the Wee1 protein, one involving phosphorylation of its C-terminal domain by the Nim1 protein and the other involving phosphorylation of its N-terminal region by a different kinase.     

Drosophila Wee1 kinase regulates Cdk1 and mitotic entry during embryogenesis

Cyclin-dependent kinases (Cdks) are the central regulators of the cell division cycle. Inhibitors of Cdks ensure proper coordination of cell cycle events and help regulate cell proliferation in the context of tissues and organs. Wee1 homologs phosphorylate a conserved tyrosine to inhibit the mitotic cyclin-dependent kinase Cdk1. Loss of Wee1 function in fission or budding yeast causes premature entry into mitosis. The importance of metazoan Wee1 homologs for timing mitosis, however, has been demonstrated only in Xenopus egg extracts and via ectopic Cdk1 activation . Here, we report that Drosophila Wee1 (dWee1) regulates Cdk1 via phosphorylation of tyrosine 15 and times mitotic entry during the cortical nuclear cycles of syncytial blastoderm embryos, which lack gap phases. Loss of maternal dwee1 leads to premature entry into mitosis, mitotic spindle defects, chromosome condensation problems, and a Chk2-dependent block of subsequent development, and then embryonic lethality. These findings modify previous models about cell cycle regulation in syncytial embryos and demonstrate that Wee1 kinases can regulate mitotic entry in vivo during metazoan development even in cycles that lack a G2 phase.     

Cell cycle-regulated phosphorylation of the human SIX1 homeodomain protein

Human SIX1 (HSIX1) is a member of the Six class of homeodomain proteins implicated in muscle, eye, head, and brain development. To further understand the role of HSIX1 in the cell cycle and cancer, we developed an HSIX1-specific antibody to study protein expression at various stages of the cell cycle. Our previous work demonstrated that HSIX1 mRNA expression increases as cells exit S phase and that overexpression of HSIX1 can attenuate a DNA damage-induced G(2) cell cycle checkpoint. Overexpression of HSIX1 mRNA was observed in 44% of primary breast cancers and 90% of metastatic lesions. Now we demonstrate that HSIX1 is a nuclear phosphoprotein that becomes hyperphosphorylated at mitosis in both MCF7 cells and in Xenopus extracts. The pattern of phosphorylation observed in mitosis is similar to that seen by treating recombinant HSIX1 with casein kinase II (CK2) in vitro. Apigenin, a selective CK2 inhibitor, diminishes interphase and mitotic phosphorylation of HSIX1. Treatment of MCF7 cells with apigenin leads to a dose-dependent arrest at the G(2)/M boundary, implicating CK2, like HSIX1, in the G(2)/M transition. HSIX1 hyperphosphorylated in vitro by CK2 loses its ability to bind the MEF3 sites of the aldolase A promoter (pM), and decreased binding to pM is observed during mitosis. Because CK2 and HSIX1 have both been implicated in cancer and in cell cycle control, we propose that HSIX1, whose activity is regulated by CK2, is a relevant target of CK2 in G(2)/M checkpoint control and that both molecules participate in the same pathway whose dysregulation leads to cancer.